Accelerated sarcolemmal phospholipid catabolism during myocardial ischemia is an important contributor to electrophysiologic dysfunction and myocyte cell death. Since plasmalogens are the predominant phospholipid constituents of sarcolemma and are selectively hydrolyzed during myocardial ischemia, the major goal of the proposed studies is the demonstration that accelerated plasmalogen catabolism during ischemia is localized to the sarcolemma and is a biochemical mediator of sarcolemmal dysfunction. To circumvent the previously insurmountable problem of assessing accelerated plasmalogen catabolism in the sarcolemmal compartment of intact ischemic hearts, electron microscopic autoradiography employing maximum-likelihood analysis will be utilized. To discriminate between phospholipase C- and D- mediated plasmalogen polar head group turnover, electrospray mass spectrometry will be exploited to quantitate 18O-labeled phosphocholine and 18O-labeled phosphatidic acid produced by cardiac myocytes that are labeled with H218O. Electrophysiologic dysfunction mediated by alterations in polar head group turnover will be assessed in voltage-clamped Sf9 cells (containing overexpressed K+ channels) and neonatal cardiac myocytes that overexpress either phospholipase C, choline phosphotransferase or ethanolamine phosphotransferase. To assess electrophysiologic alterations mediated by ischemia-activated calcium-independent PLA2, patch-clamped cardiac myocytes will be perfused with purified calcium-independent PLA2 and the specificity of electrophysiologic alterations will be determined utilizing a specific mechanism-based inhibitor of calcium-independent PLA2 (HELSS). The physiologic sequelae of accelerated plasmalogen catabolism also will be assessed by molecular characterization of myocardial lysoplasmenylcholine-activated protein kinase and by identifying specific myocardial protein kinase C isozymes that are activated by alkenyl-acyl glycerol during myocardial ischemia. Taken together, the proposed research represents a multidisciplinary state-of-the-art approach to facilitate the direct demonstration of accelerated plasmalogen catabolism as an important mediator of the pathophysiologic sequelae of myocardial ischemia.